Water-soluble vitamins separation

Phenomenex, Fat and water soluble vitamin separations, Resolution times, Summer (1993). 

Fig. 8-87. Analysis of water-soluble vitamins. - Separator column Spherisorb ODS 2 (5 pm) eluent (A) 0.1 mol/L KOAc (pH 4.2 with HOAc), (B) water/methanol/acetonitrile (50 10 40 v/v/v) gradient linear, 6% B in 30 min to 100% B flow rate 2 mL/min detection UV (254 nm) injection volume 50 pL solute concentrations 5 nmol each of ascorbic acid (1), nicotinic acid (2), thiamine (3), pyridoxine (4), nicotinic add amide (5), p-aminobenzoic add (6), cyanocobala-mine (7), and riboflavine (8).

Fernandez,M., R. Forteza,andV. Cerda. 2012. Multisyringechromatography(MSC) An effective and low cost tool for water-soluble vitamin separation. Anal. Lett. 45 2637-2647. 

Ion-pairing reagents are detergent-like molecules added to the mobile phase to provide additional retention or selectivity for the analytes with opposite charge. Long-chain alkyl sulfonates are commonly used for the separation of water-soluble basic analytes as shown in Figure 16 in the analysis of water-soluble vitamins (WSV). Hexanesulfonate binds with... 

Water-soluble vitamins in formulations have been determined by use of ion-pair chromatography. The vitamins include several B vitamins as well as niacin, folic acid, and ascorbic acid (565). Vitamins D and Da were rapidly separated on reverse phase columns (247) as are vitamins A, D, and E in multivitamin tablets (564). Addition of silver ions to the mobile phase has been shown to increase the flexibility inherent in RPC by complexing with the unsaturated bonds and thereby decreasing the retention factor. This effect is also observed with other unsaturated drug molecules including steroids (247). Vitamin A and related compounds have... 

Capillary zone electrophoresis is a powerful tool for the separation of water-soluble vitamins, such as nicotinic acid and vitamin C, with high-pH borate or phosphate buffers. Most simultaneous separations have been performed for fat-soluble vitamins, such as vitamins A and E, by MEKC. Here, organic... 

A large number of methods have been developed for analysis of water-soluble vitamins simultaneously in pharmaceutical products (like multivitamin tablet supplements). In fact, for these products no particular sample preparations are required and the high concentrations simplify the detection, enabling the use of UV [636]. The use of MS is also reported [637]. As well, Moreno and Salvado [638] reports also the use of a unique SPE cartridge (C18) for separating fat-soluble and water-soluble vitamins, which are, then analyzed using different chromatographic systems. 

Recently, the use of a novel stationary phase (p-tcrt-butyl-calix[8]arene-bonded silica gel, CABS) has been reported for the separation of six water-soluble vitamins (Bj, B2, Bg, Bj, B12, and C) by using isocratic elution with methanol/acetate buffer [639]. 

The water-soluble vitamins niacinamide (a neutral compound), riboflavin (a neutral compound), niacin (an anion), and thiamine (a cation) were separated by micellar electrokinetic chromatography in 15 mM borate buffer (pH 8.0) with 50 mM sodium dodecyl sulfate. The migration times were niacinamide (8.1 min), riboflavin (13.0 min), niacin (14.3 min), and thiamine (21.9 min). What would the order have been in the absence of sodium dodecyl sulfate Which compound is most soluble in the micelles ... 

The water-soluble vitamins are a highly diverse group of compounds with differing physicochemical properties. A single vitamin generally consists of several vitamers, or chemical species, each of which exhibits the same biological activity in vivo. Individual vitamers with the same biological functions often exhibit vastly different physicochemical properties. This necessitates unique extraction and separation procedures for each vitamin. As a result, each vitamin is considered individually in this chapter. A section on methods that determine multiple vitamins simultaneously is also included. 

The first grouping is a mix of fat-soluble compounds that function as hormones, co-factors, and membrane components. Fat-soluble vitamins separate on a Cis column in 80% acetonitrile/water and are usually detected at UV, 280 nm, or with fluorescence. Triglycerides are slightly less nonpolar than fat-soluble vitamins and require 60% acetonitrile/water to run on Ci8. They have poor extinction coefficients, and detection at UV, 220 nm, competes with refractive index detection in sensitivity. A phenyl column run in 50%... 

First, mobile phase and column we saw that most of the small molecule separations could be made on a C18 column in An/buffer water, with the exception of charged molecules and carbohydrates, which are too water soluble. We saw a range of polarity from fat-soluble vitamins, steroids, triglycerides, chlorinated pesticides eluting in 60-80% An/water, to carbamate, phosphate pesticides, anticonvulsants, antidepressants at 40-50% An/water, to nucleosides, nucleotides, aspirin, and water-soluble vitamins at 5-10% An/water. If you know something about the compound s structure or its solubility, you have a good clue as to what mobile phase can be used for its separation. 

Among more complex macrocycles, Li et al. [47-52] reported the preparation and characterization of stationary phases incorporating calixarenes or calix-crowns bonded to silica. With individual columns, high selectivity was observed in the separation of alkylated aromatics, aromatic carboxylic acids, sulfonamides, nucleosides, and water-soluble vitamins. In other work, Sokoliess et al. [53] have characterized calixarene- and resorcinarene-bonded stationary phases similar to those described in the previous section of this chapter. And Huai et al. [54] used an end-capped p-tert-butyl-calix[4]arene-bonded silica phase for HPLC separation of a number of organic compounds. Resorcinarenes have also found application in GC. [55-57] Recently, exotic macrocycles have been used in capillary electrochromatography, as reported by Gong et al. [58]... 

The separation of water-soluble vitamins, including vitamin Bg, on ion-exchange paper has been described by Klotz and Huettenrauch, (101) using Amberlite SB-2, Amberlite WB-2, and Amberllte WA-2. 

Several methods have been reported for the separation of water soluble vitamins including vitamin Bg. Some of these systems are described as follows ... 

M. C. Gennaro, Separation of water-soluble vitamins by reversed-phase ion-interaction-reagent high-performance liquid chromatography Application to multivitamin pharmaceuticals, J. Chromatogr. Sci., 29 410(1991). 

Figure 5.8 Separation of eleven water-soluble vitamins by MECC. Peaks 1, pyridoxamine 2, nicotinamide 3 pyridoxal 4, vitamin B6 5, vitamin B2 6, vitamin B12 7, vitamin B2 phosphate 8, pyridoxamine 5 -phosphate 9, niacin 10, vitamin Bi 11, pyridoxal 5 -phosphate. Conditions buffer, 50 mM SDS in 20 mAf phosphate-borate buffer, pH 9.0 applied voltage, 20 kV detection, UV absorbance at 210 nm. (Reprinted from Ref. 20 with permission.)...

SHW has also been apphed to the separation of nutraceuticals, natural products, and biochemicals, including the water-soluble vitamins, thiamine, riboflavin, and pyidoxine (Table 18-3) without significant thermal degradation. 

The technique can be used to separate a wide range of biologically important molecules such as amino acids, peptides, nudeobases, nudeosides and nucleotides, water-soluble vitamins and steroid hormones etc. It is being increasingly used in the analysis of pharmaceuticals and their metabolic products. 

The potential of PBI LC-MS in the analysis of various vitamins was explored by Careri et al. [99-100]. The fat-soluble vitamins A, D, and E were analysed in food and multivitamin preparations [99]. Absolute detection limits in SIM mode were 0.6-25 ng after fast leversed-phase separation using a 97% aqueous methanol as mobile phase. Mass spectra in El, positive-ion and negative-ion Cl were obtained and discussed. The mass-spectral and quantitative performance of PBI LC-MS in the analysis of eleven water-soluble vitamins was also explored [100]. Detection limits were determined in SIM mode under positive-ion Cl, and were below 15 ng for ascorbic acid, nicotinamide, nicotinic acid, and pyridoxal, around 100 ng for dehydroascorbic acid, panthothenic acid, and thiamine, and above 200 ng for biotin, pyridoxamime, and pyridoxine. Riboflavine was not detected. 

Fig. 1 CCC separation of water-soluble vitamins by cross-axis CPC. Experimental conditions apparatus, cross-axis CPC equipped with a pair of eccentric coil assemblies, 1 mm ID and 26.5 mL capacity sample. (A) thiamine nitrate (2.5 mg)-1-riboflavin (1.5 mg)-I-nicotinamide (2.5 mg) (B) riboflavin sodium phosphate (2.5 mg)-1-nicotinamide (2.5 mg) and (C) thiamine nitrate (2.8 mg) + pyridoxine hydrochloride (4.0 mg)-1-nicotinamide (3.0 mg) solvent system (A) and (B) 1-butanol/aqueous 0.15 M monobasic potassium phosphate (1 1) and (C) 1-butanol/ethanol/aqueous 0.15 M monobasic potassium phosphate (8 3 8) mobile phase lower phase flow rate 0.4 mL/min revolution 800 rpm. SF = solvent front.

In order to improve the separation of each water-soluble vitamin by cross-axis CPC, three ion-pair reagents were added to the 1-butanol/aqueous 0.15 M monobasic potassium phosphate system. The K values of... 

Shinomiya, K. Yoshida, K. Kabasawa, Y. Ito, Y. Countercurrent chromatographic separation of water-soluble vitamins by cross-axis coil planet centrifuge using an ion-pair reagent with polar two-phase solvent system. J. Liq. Chromatogr. Relat. Technol. 2001, 24, 2615. 

Water-soluble vitamins (vitamin Bi, vitamin Bg, vitamin B12, and vitamin C) in Kombucha drink (a curative liquor) were separated by TLC on silica gel plates with water as the solvent. The plates were visually examined under UV light at 254- and 366-nm wave-g lengths. The four vitamins were identified and determinec by comparing the Rf values with the reference values (vitamin Bi, 0.21 vitamin Bg, 0.73 vitamin B12,0.34 anc vitamin C, 0.96). ... 

Perhaps it is more than happenstance that vitamin C is separated by name from the other water soluble B vitamins. Historically, this separation was the result of the normal procedure of labeling unknown dietary factors, as C was diflFerentiated from B— which later was shown to be a complex group rather than an individual, water-soluble vitamin. It was... 

When you read descriptions of what vitamins and minerals do in the body, including my own descriptions of their functions and activities, the benefits described almost always include enzyme activities without actually giving credit to the enzymes. Why Because all water-soluble vitamins and many minerals and trace elements inside us act as coenzymes or are part of the structure of enzymes, and often you can t separate what the enzyme does from what its trace element and vitamin components do. Deficiencies of trace elements in the diet can prevent enzymes needing those elements from doing their jobs. 

Jegle [41] described the separation of water soluble vitamins by CZE, and gave an example of analysis from a commercial vitamin preparation (Figure 7). Lambert, et al. compared the analysis of B12 and analogues by HPLC and CZE. The CZE method was tested on multi-vitamin preparations [42]. Ma, et al., used CZE and laser induced fluorescence for the fast microassay of vitamin A in serum samples [43]. 

L Fotsing, B Boulanger, P Chiap, M Fillet, P Hubert, J Crommen. Multivariate optimization approach for the separation of water-soluble vitamins and related compounds by capillary electrophoresis. Biomed Chromatogr 14 10-11, 2000. 

Water-soluble vitamins are also known as the B Complex because it was originally considered as one vitamin. It was later discovered that these are separate vitamins. The following are water-soluble vitamins. 

Vitamin Bg exists as six separate forms in the pyridine group of water-soluble vitamins. The common forms are pyridoxal and pyri-doxamine together with their corresponding phosphate esters and pyridoxine forms. These compounds function as cofactors in a wide variety of enzyme reactions, but most notably in the transamination reaction of amino acid biosynthetic pathways. Extraction of this group of vitamins can be performed by the same methods as those described for the B2 vitamins (Section 11.8.3.3). 

---